Abstract. Cytosolic and endosomal DNA sensing pathways are known to play a critical role in host defense against microbial pathogens. The same pattern recognition receptors also detect endogenous ligands and thereby promote the onset and progression of autoimmune and autoinflammatory diseases. For example, endosomal TLRs contribute to the pathogenesis of Systemic Lupus Erythematosis (SLE), in part by promoting the production of type I IFNs. Cytosol DNA can be detected by a variety of receptors, including cGAS, which in turn generates an unusual cyclic dinucleotide that activates pathways downstream of STING. Overactivation of STING, either by loss of nuclease activity or STING gain-of-function mutations, also drives a strong type I IFN response now associated with diseases such as Aicardi-Goutieres Syndrome. We have explored the potential crosstalk between cytosolic and endosomal sensors in murine SLE. Quite unexpectedly, we found that STING-deficient SLE-prone mice developed more severe, not less severe, clinical disease. These obser- vations point to a novel role for STING in the negative regulation of TLR-driven systemic autoimmunity. Our preliminary studies have led us to propose that constitutive activation of the STING pathway in hemato- poietic cells limits the inflammatory response of myeloid cells to TLR ligands, promotes the production of negative regulators of immune activation such as A20 and immunomodulatory enzymes such as Indoleamine-pyrrole 2,3-dioxygenase (IDO), and contributes to B cell tolerance induction; the nucleotidyl transferase DNA sensor cGAS acts upstream to recognize host DNA and regulate these STING-mediated effects. We will validate and explore this hypothesis through the following specific aims: (1) identify the cell types directly activated as a result of STING-deficiency; (2) explore the molecular mechanisms responsible for STING-mediated suppression of TLR-driven inflammation; and (3) determine the role of cGAS in the negative regulatory role of STING. To precisely compare STING-sufficient and STING-deficient macrophages, dendritic cells and B cells in models of SLE, we will utilize autoimmune-prone CD45 and Igh allelically distinct mixed bone marrow chimeras.This strategy will preclude any potential confounding factors arising from subclinical infection, changes in microbiome, or effects of disease driven inflammation. SLE is a complex chronic systemic autoimmune disease that afflicts over 1.5 million Americans. Current treatments involve immuno-suppressive regimens associated with debilitating adverse side effects. Attempts to develop safe and efficient therapies that block TLR activation have been stymied by the relative short in vivo half lives of known inhibitors and the potential dangerous outcome of complete MyD88 blockade. Better understanding of the natural regulators of the disease process will provide major insights toward the design of more disease-specific therapeutic options and also avoid the use of STING antagonists that could trigger unanticipated dangerous outcomes. Therefore, the mechanisms we are exploring are highly relevant to human disease and key to future targeted therapies.